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Fig. 1. Structure and developmental expression of the
leiomodin (Lmod) and tropomodulin (Tmod) gene families.
(A) leiomodin (Lmod1–3) and tropomodulin (Tmod1–4) proteins are
structurally related, yet possess unique domains. Lmods and Tmods
share actin-binding (A1, A2), tropomyosin-binding (TM1) and
leucine-rich-repeat (LRR) domains. A second tropomyosin-binding
domain (TM2) is unique to the Tmods. Lmod proteins have a Cterminal
extension that contains a third actin binding/WH2 domain
(WH2/A3). (B–I) Lmod (C–E) and Tmod (F–I) family gene expression
was analyzed during Xenopus embryonic development using in situ
hybridization. All embryos are positioned with heads to the left.
Expression patterns are shown at the early tailbud stage (st30),
during the early stages of myofibrillogenesis. Earlier expression
during the neurula stage (st15) is presented in supplementary
material Fig. S1. Expression of the definitive striated muscle marker
cardiac actin, actna1 (B), was used to indicate cardiac and skeletal
muscle tissue in the embryo. Arrows indicate developing skeletal
muscle and heart tissues. Only lmod3 (E) and tmod4 (I) are
expressed at high levels in developing skeletal muscle.
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Fig. 2. Tmod4 and Lmod3 localize to the M-line during
Xenopus skeletal myofibrillogenesis. The somite regions of
Xenopus embryos were sectioned and protein distribution was
examined using Texas-Red-conjugated phalloidin and antibodies
against a-actinin, Tmod4 or Lmod3. (A–L) Localization of Tmod4
protein during skeletal muscle myofibrillogenesis. (A–D) At st20,
continuous actin filament staining was visible (A), with no periodic
localization of a-actinin (B) or Tmod4 staining (C). (E–H) At st24,
diffuse striations were visible in a small number of actin filaments
(E). In these striated regions, narrow bands of a-actinin (F,
arrowheads) and Tmod4 staining (G, arrows) were visible. (I–L) At
st34, broad sharply striated (i.e. highly organized) actin filaments
(I) were observed throughout the skeletal muscle tissue. Sharp
regions of a-actinin (J, arrowheads) and Tmod4 staining (K) were
visible at the Z-disc and M-line regions, respectively. (M–X)
Localization of Lmod3 protein during skeletal muscle
myofibrillogenesis. (M–P) At st20, continuous actin filament
staining was visible, but no localization of a-actinin (N) or Lmod3
(O) was observed. (Q–T) At st24, some regions of developing
muscle showed narrow diffusely striated actin filaments (Q). In
these regions, narrow bands of a-actinin (R, arrowheads) and
Lmod3 staining (S, arrows) were visible. (U–X) At st34, broad
sharply striated actin filaments were visible throughout the
developing somites. Staining for a-actinin (V, arrowheads) and
Lmod3 (W, arrows) was detected at the Z-line and M-line regions,
respectively. We conclude that both Tmod4 and Lmod3 proteins
are present at the M-line region of the sarcomere from the earliest
stages of myofibril assembly. Scale bar: 5 mm.
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Fig. 3. Tmod4 is essential for skeletal muscle myofibril
assembly. (A) Embryos treated with control MO (upper panel)
and tmod4 MO (lower panel) develop normally. (B) Protein
blots demonstrate that the anti-Tmod antibody specifically
recognizes Xenopus Tmod4 protein (lane labeled GFP–
Tmod4) and that the tmod4 MO effectively blocked translation
of Tmod4 protein. (C–R) Loss of Tmod4 protein resulted in
disruption of sarcomere assembly. (G–J) Sections through
developing muscle tissue of embryos depleted of Tmod4
protein show severely disrupted sarcomeres compared with
control embryos at the same developmental stage
(C–F). (O–R) Localization of other sarcomeric components,
including the N-terminal region of titin (P) and myosin heavy
chain (Q) was also severely disorganized compared with that
of controls (K–N). (S–Aa) The specificity of the Tmod4
knockdown was demonstrated by rescue experiments. In
these studies, tmod4 MO was co-injected into the Xenopus
embryo with mRNA encoding GFP–Tmod4. Organization of
both thin filament (S–V) and other sarcomeric proteins
(X,Y) was comparable to that of unmanipulated controls
(C–F). All fluorescent images (C–Z) are presented at the same
scale. Scale bar: 5 mm. Quantification of experimental results
(Aa) showed that depletion of Tmod4 protein in st34 embryos
resulted in a dramatic reduction in the percentage of correctly
structured sarcomeres (,5.4% of fibers showing M-line gaps
following phalloidin staining compared to 62.5% in controls).
Addition of mRNA encoding GFP–Tmod4, together with the
MO, resulted in rescue of sarcomere structure. Data show the
mean6s.e.m.; ***P,0.001 (x2 analysis).
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Fig. 4. Lmod3 is essential for skeletal myofibril assembly.
(A) Embryos treated with control MO (upper panel) and lmod3
MO (lower panel) develop normally. (B) Protein blots
demonstrate that the anti-Lmod antibody specifically
recognizes Xenopus Lmod3 protein (lane labeled Myc–
Lmod3) and that the lmod3 MO effectively blocked translation
of Lmod3 protein. (C–R) Loss of Lmod3 protein resulted in
disruption of sarcomere assembly. (G–J) Sections through
developing muscle tissue in embryos depleted of Lmod3
protein show severely disrupted sarcomere structure
compared with that of control embryos of the same
developmental stage (C–F). (O–R) The localization of other
sarcomeric components, including titin (P) and myosin (Q) was
also severely disorganized compared with that of controls
(K–N). (S–Aa) The specificity of the Lmod3 knockdown was
demonstrated by rescue experiments. lmod3 MO was coinjected
into the Xenopus embryo with mRNA encoding Myc–
Lmod3. Organization of thin filament (S–V) and other
sarcomeric proteins (X,Y) was comparable to that of
unmanipulated controls (C–F). Scale bar: 5 mm. Quantification
of results (Aa) showed that depletion of Lmod3 protein in
st34 embryos resulted in a dramatic reduction in the
percentage of correctly structured sarcomeres (70.2% in
control MO-treated compared to 23.6% in lmod3 MO-treated).
The addition of mRNA encoding Myc–Lmod3 together with the
lmod3 MO restored sarcomere structure to control levels. Data
show the mean6s.e.m.; ***P,0.001 (x2 analysis).
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Fig. 5. Lmod3 and Tmod4 display largely equivalent
functions during skeletal myofibril assembly. (A–L) In
these studies, tmod4 MO was co-injected into the Xenopus
embryo along with mRNA encoding Myc–Lmod3. (E–L) When
Myc–Lmod3 was added to embryos depleted of Tmod4,
sarcomere assembly was significantly rescued compared with
that of embryos depleted of Tmod4 alone (A–D). Note that in
regions showing clear striated organization, Myc–Lmod3 was
abundant at the M-line (F,J), whereas Tmod4 was not
detected (G). The structure of other sarcomeric proteins,
assayed by localization of myosin (K) was also restored.
(M–X) In these studies, lmod3 MO was co-injected into the
Xenopus embryo along with mRNA encoding GFP–Tmod4
(Q–X). When GFP–Tmod4 was added to embryos depleted of
Lmod3, sarcomere assembly was significantly rescued
compared with that of embryos depleted of Lmod3 alone (M–
P). Note that in regions showing mature striated organization,
GFP–Tmod4 was abundant at the M-line (R,V), whereas
Lmod3 was not detected (S). The structure of other
sarcomeric proteins, assayed by the localization of myosin
(W) was also restored. Scale bar: 5 mm. (Y,Z) Quantification of
experimental results showed that the addition of Myc–Lmod3
to embryos depleted of Tmod4 resulted in a significant
increase in the percentage of sarcomeres displaying normal
structure compared with that of Tmod4-depleted embryos
(5.6-fold increase). Similarly, addition of GFP–Tmod4 to
embryos depleted of Lmod3 resulted in a dramatic rescue of
sarcomere structure compared with that observed in Lmod3-
depleted muscle (2.9-fold). Data show the mean6s.e.m.;
***P,0.001 (x2 analysis).
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Fig. S1. Developmental expression of Tmod and Lmod family proteins at the
neurula stage prior to skeletal muscle assembly. Tmod (B-D) and Lmod (E-H)
family gene expression was analyzed using in situ hybridization. All embryos are
positioned with heads to the left. Expression patterns were analyzed at the neurula
stage (st15), before the onset of myofibrillogenesis. Expression of the striated muscle
marker, cardiac actin, actna1 (A) was used to indicate skeletal muscle tissue in the
embryo. Arrows indicate developing skeletal muscle in the somites. Only lmod3 (D) and
tmod4 (H) are expressed at high levels in developing skeletal muscle.
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Fig. S2. Expression of endogenous lmod and tmod family genes is not
altered by tmod4 MO treatment. tmod (A-H) and lmod (I-N) family gene expression
was analyzed in control MO- (left panels) and tmod4 MO- (right panels) treated embryos
using in situ hybridization. All embryos were assayed at tailbud stage (st34), the same
stage that myofibril structure was analyzed. No appreciable alteration of gene expression
is observed following treatment with tmod4 MO.
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Fig. S3. Localization of tagged Tmod4 and Lmod3 proteins. (A-D) At the
levels used for rescue studies, GFP-Tmod4 did not alter sarcomere or thin filament
dimensions or localization of Lmod3 at the M-line. Embryos were microinjected with
900pg of GFP-Tmod4 mRNA and assayed for myofibril organization at stage 34.
Endogenous Lmod3 localized to the M-line in the presence of additional GFP-Tmod4
protein. (E-H) At the levels used for rescue studies, myc-Lmod3 did not alter sarcomere
or thin filament dimensions or localization of Tmod4 at the M-line. Embryos were
microinjected with 900pg of mRNA encoding myc-Lmod3 and assayed at stage 34.
Endogenous Tmod4 localized to the M-line in the presence of additional myc-Lmod3
protein. In all panels, arrows indicate localization of Tmod4 at the M-line, and
arrowheads indicate Lmod3 localization, co-localizing with Tmod4 at the M-line. Scale
bar is 5 microns.
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Fig. S4. Truncated versions of Lmod3 protein, lacking the C-terminal
extension, do not incorporate into developing myofibrils and cannot rescue
sarcomere structure in knockdown experiments. Two separate Lmod3
truncations (Lmod3d1 and Lmod3d2) were generated. Lmod3d1 is truncated at a
position exactly equivalent to the C-terminus of Tmod4 and Lmod3d2 is 11 aa longer.
(A-H). In contrast to full length Lmod3 (Fig. S4), truncated forms of Lmod3 did not
detectably incorporate into developing sarcomeres. (I-T). Myofibrillogenesis was
severely disrupted by depletion of Tmod4 protein synthesis using tmod4 MO, but this
could be rescued by addition of either GFP-Tmod4 or GFP-Lmod3 proteins. (U-Ab). In
contrast to full-length GFP-Lmod3 (Q-T) neither of the truncated forms of Lmod3 was
sufficient to rescue sarcomere structure. (Ac). Western blot, using anti-GFP antibody,
showing equivalent expression of GFP-Lmod3 and GFP-Lmod3 truncations in injected
embryos.
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lmod1 (leiomodin 1 (smooth muscle)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.
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lmod1 (leiomodin 1 (smooth muscle)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.
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lmod2 (leiomodin 2 (cardiac)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.
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lmod2 (leiomodin 2 (cardiac)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.
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lmod3 (leiomodin 3 (fetal)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.
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lmod3 (leiomodin 3 (fetal)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.
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tmod1 (tropomodulin 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.
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tmod1 (tropomodulin 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.
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tmod2 (tropomodulin 2 (neuronal)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.
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tmod2 (tropomodulin 2 (neuronal)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.
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tmod3 (tropomodulin 3 (ubiquitous)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.
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tmod3 (tropomodulin 3 (ubiquitous)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.
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tmod4 (tropomodulin 4 (muscle)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.
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actc1 (actin, alpha, cardiac muscle 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.
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actc1 (actin, alpha, cardiac muscle 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.
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